Pluripotent hematopoietic stem cells are activated in the bone marrow to proliferate and differentiate into mature megakaryocytes, each of which is capable of releasing up to several thousand functional platelets in response to biological demand. Development of the stem cell proceeds by stages broadly corresponding to proliferation of progenitor cells, and differentiation of late progenitor and early precursor cells into mature megakaryocytes. Although regulation of this developmental process (megakaryocytopoiesis) is of substantial clinical interest for its potential application to disorders characterized by abnormal platelet production, endogenous factors responsible for stimulating or inhibiting proliferation and differentiation of megakaryocyte progenitor/precursor cells have not been thoroughly elaborated.
Thrombocytosis is a condition marked by the absolute increase in the number of circulating platelets. In some cases the elevation is acute and transient; in others it is chronic and persistent. The term "reactive thrombocytosis" has been commonly applied to define the concept that these patients have increased circulating platelet numbers in response to some underlying disease. This is in contrast to the condition where an autonomous drive to platelet production exists, commonly termed "thrombocythemia".
Reactive thrombocytosis may appear and persist as a result of chronic blood loss with iron deficiency, chronic inflammatory disease, chronic infectious disease, cancer and hemolytic anemia.
Primary thrombocythemia, also known as essential thrombocythemia, is an autonomous clonal proliferation of a pluripotent hematopoietic stem cell that results in an absolute increase in the number of circulating platelets. It shares several clinical features with other myeloproliferative disorders, most notably frequent bleeding and thrombotic lesions that represent major causes of morbidity and mortality.
Inhibitory factors capable of clinically significant megakaryocyte suppression have not been well-characterized. For example, both immunocytes and transforming growth factor-.beta. (TGF-.beta.) have been studied as potential inhibitors of megakaryocytopoiesis, with inconclusive results (see, e.g., Blood 67, 479-483 and Blood 68, 619-626, (1986) Additionally, autoregulation via negative feedback mechanisms involving megakaryocyte products, including platelet-secreted 12-17kD glycoprotein, has been reported (J. Cell Physiol. 130, 361-368, (1987)). Platelet factor 4 and a synthetic C-terminal peptide have been shown to be capable of inhibiting megakaryocytopoiesis (Gewirtz et al., J. Clin Invest. 83, 1477-1486 (1989)). It has also been suggested that interferon-.beta. and interferon-.gamma. may have a role in regulating megakaryocyte colony formation (Ganser et al., Blood 70, 1173-1179 (1987); Chott et al., Br. J. Haematol. 74, 10-16 (1990)). While interferon-.alpha. has been used to lower platelet counts in patients with primary thrombocythemia and thrombocytosis associated with other types of malignant lesions, only approximately about 50% of patients achieve a stable state of remission. Moreover, on cessation of interferon therapy, recurrence of clinical and laboratory findings is usual (Gisslinger et al., Lancet 1, 634-637 (1989)).
While the potential utility of negative autocrine regulators or other megakaryocytopoiesis inhibitors in the clinical treatment of disorders characterized by excessively high platelet counts is apparent, none of the heretofore postulated inhibitors has so far proved useful in such applications.
Cytoreducive chemotherapeutic agents such as alkylating agents, radiophosporous and antimetabolites have been used to reduce platelet numbers. Most have leukemogenic potential. Their use has largely been abandoned in favor of hydroxyurea. However, hydroxyurea should at best be considered an agent with uncertain carcinogenic potential because at least one case of primary thrombocythemia conversion to acute leukemia has been linked to hydroxyurea therapy (Anker-Lugtenberg et al., Am. J. Hematol. 33:152 (1990)).
Anagrelide, a member of the imidazo (2, 1-b) quinazolin-2-one series, is an investigational drug which has been recently proposed for the treatment of thrombocytosis. Anagrelide has been shown to be capable of controlling platelet counts in most patients suffering from essential thrombocythemia as a consequence of an underlying myeloproliferative disorder. Suppression of platelet counts by anagralide appears to be selective relative to changes in white blood cell count and hemoglobin. However, the drug's potent effect on inhibiting platelet activation requires further study.
Macrophage inflammatory protein-1 (MIP-1) is a heparinbinding protein secreted by macrophages in response to lipopolysaccharide stimulation. MIP-1 is a major secretion product from stimulated macrophages, comprising about 2% of proteins secreted by endotoxin-stimulated cells. MIP-1 causes a local inflammatory response in mice and induces superoxide production in human neutrophils in vitro. It is also mildly chemokinetic for human neutrophils. MIP-1 is composed of two distinct peptides, MIP-1.alpha. and MIP-1.beta..
Macrophage inflammatory protein-2 (MIP-2), is another heparin-binding protein secreted by lipopolysaccharide-stimulated macrophages. It comprises about 0.5% of the proteins secreted by stimulated macrophages. Like MIP-1, MIP-2 has been shown to elicit a local inflammatory response when injected subcutaneously into mice. It has potent chemotactic activity for human polymorphonucleocytes. Also like MIP-1, MIP-2 is composed of two distinct peptides, MIP-2.alpha. and MIP-2.beta..
The activity of MIP-1 and MIP-2 in inhibiting megakaryocytopoiesis was unknown prior to the invention hereinafter described.